Production of proteins in host cells

ABSTRACT

Method of increasing protein production in a host cell where the nucleic acid encoding the protein has an A and/or T rich region in an exon. The method comprising lowering the amount of A and/or T rich region and transfecting a host cell with the altered nucleic acid and obtaining expression of the nucleic acid. The invention also relates to the recombinant nucleic acid constructs, purified protein preparations and pharmaceutical compositions produced as well as methods of treatment utilizing the purified protein preparation.

FIELD OF THE INVENTION

The present invention relates generally to improved production ofproteins in host cells. In particular it relates to improved productionof proteins which are required for therapeutic or reagent applicationsand which are difficult to obtain in sufficient quantities. Theinvention also relates to a method of producing recombinant nucleic acidmolecules which encode said proteins, to the recombinant nucleic acidmolecules and recombinant constructs comprising the same and to theproteins produced thereby. Of particular interest are nucleic acidmolecules encoding hormones, cytokines, growth factors, receptors andtheir ligands and cell surface proteins including complement regulatingproteins.

BACKGROUND OF THE INVENTION

Although many genes and cDNAs encoding useful proteins have beenisolated, a major problem facing scientists is the production ofsufficient amounts of recombinant proteins for further study and fordiagnostic or therapeutic applications. This is particularly so wherecommercial production of the protein is desired. In particular theproduction of hormones, cytokines, growth factors, receptors and theirligands and other proteins by recombinant means has become a majorendeavour.

By way of example the cell surface molecule, CD46 (also known asmembrane cofactor or MCP) has not been expressed in eukaryotic hosts aswell as other cDNA constructs using a variety of expression vectors.Similar concerns have arisen about CD55 (DAF) and CD35 (CR1) and factorH which like CD46, are members of the family of proteins calledRegulators of Complement Activation (RCA).

Regulators of Complement Activation control the amplification of thecomplement cascade. Each of the members of this family share structuralcharacteristics, principally the ˜60-65 amino acid Short ConsensusRepeat (SCR) modules, which are responsible for complement binding andregulatory functions. Genes encoding the family members are linked andmap to chromosome 1q3.2.

The biology of these molecules is of interest in inflammation andhomeostasis. However, there is currently a further application for themin the potential use of porcine organs or other organs for grafting tohumans. Such xenogenic organs undergo antibody and complement mediatedhyperacute rejection.

A limitation to studies of CD46 and other RCAs has been that ontransfection with constructs encoding the recombinant proteins in eithertransient or stable systems very few cells have been transfected andthose that are express low amounts of protein. Indeed, in the inventors'laboratory and others, sophisticated cell sorting, cloning and selectionprocedures have been necessary to isolate appropriate cells. This, inparticular, has created problems with the analysis of recombinant CD46,its physiological role and value as a therapeutic agent.

In work leading up to the present invention the inventors havesurprisingly found that alterations in a nucleic acid encoding CD46 ledto high levels of CD46 production in a transfected eukarotic host.Protein production levels of up to 8 times that obtained from thewildtype gene for CD46 have been observed. These alterations comprisedreducing the amount or proportion of adenine (A) and/or thymine (T) inan A and/or T rich region in the nucleic acid.

SUMMARY OF THE INVENTION

The present invention provides a method of increasing or improvingmammalian protein production, without demonstrably altering RNAstability, in a host cell where a nucleic acid encoding the mammalianprotein has an A and/or T rich region present in an exon, said methodcomprising altering the nucleic acid by reducing or lowering the amountof A and/or T in said region and transfecting a host cell with saidnucleic acid and, under appropriate conditions, obtaining expression ofsaid nucleic acid.

In another aspect the present invention provides a method for enhancingproduction of RCA proteins in a host cell where a nucleic acid encodingan RCA has an A and/or T rich region present in one or more of its exonssaid method comprising altering the nucleic acid by lowering the amountof A and/or T in said A and/or T rich region, transfecting a host cellwith said nucleic acid and, under appropriate conditions, obtainingexpression of said nucleic acid.

In another aspect the present invention relates to a method ofincreasing production of an RCA protein in a host cell comprisingaltering a nucleic acid encoding the RCA by deleting a SCR module whichcontains an AT rich region, or a part thereof, transfecting a host cellwith said altered nucleic acid and under appropriate conditionsobtaining expression of said nucleic acid.

The present invention also relates to a recombinant nucleic acidconstruct which is capable of increased production of a mammalianprotein in a host cell, without demonstrably altering RNA stability,wherein the amount of A and/or T in one or more A and/or T rich regionspresent in one or more exons of the nucleic acid construct has beenreduced.

The present invention also relates to the proteins produced by themethods of the invention, to proteins encoded by the constructs of theinvention, pharmaceutical compositions, host cells transfected with theconstructs and transgenic animals expressing the proteins.

The present invention also relates to a method of producing an alteredgene encoding a protein wherein said altered gene is capable ofincreased production of the protein said method comprising altering agene with one or more A and/or T rich regions present in one or moreexons by reducing or lowering the amount of A and/or T in said regions.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Sequence of CD46 constructs in the mutated SCR2 and SCR3regions. Numbering of the sequence is based on the cDNA clone pm5.1.(Purcell et al., 1991) and extends from the end of SCR1 at nucleotide303 (nt303) to the beginning of SCR3 (nt564). SCR junctions are shown.The sequence for the construct wtSCR (equivalent to pm5.1) is shown infull with the corresponding single letter code amino acid sequence givenabove it. Nucleotides which have been substituted in any of theconstructs are underlined. For the subSCR2, subSCR3 and subSCR2+3constructs the sequence matching wtSCR is shown with a dot (.) and thesubstituted nucleotide is indicated. The deletion mutant construct is asfor the other constructs except that the SCR2 deletion is indicated byblanks. The wild type SCR amino acid sequence corresponds to SEQ IDNO:6. The wild type SCR nucleotide sequence corresponds to positions280-531 of SEQ ID NO:1. The subSCR2 nucleotide sequence corresponds topositions 280-531 of SEQ ID NO:2. The subSCR3 nucleotide sequencecorresponds to positions 280-531 of SEQ ID NO:3. The subSCR2+3nucleotide sequence corresponds to positions 280-531 of SEQ ID NO:4. ThedeISCR2/subSCR3 nucleotide sequence corresponds to positions 280-342 ofSEQ ID NO:5.

FIG. 2A-2E. Flow cytometry of COS cells after transient transfectionwith wildtype and mutant constructs. Flow cytometry was performed asdescribed in Materials and Methods. All cells were stained with CD46 mAbE4.3. Panel A shows mock transfected COS cells. Panel B shows profilesfor wtSCR transfected cells (---) overlayed with subSCR2 transfectedcells (----); panel C shows wtSCR(---) overlayed with subSCR3 (----)panel D shows wtSCR (---) overlayed with subSCR2+3 (----); panel E showswtSCF (---) overlayed with delSCR2/subSCR3(----). The mutated constructsgenerated a profile shifted to the right in each case.

FIG. 3. Western blot analysis of COS cell lysates transfected with CD46constructs (see Materials and Methods). Lanes contain 40 μl of adilution of cell lysate at 5×10⁶ cells per ml. From left to right lanescontain transfectant WOP-5.10L (CD46 BC2) at 1/5 dilution; COS celllysate transfected with no DNA at 1/10 dilution and 1/50 dilution COScell lysate transfected with wtSCR2 at 1/10 and 1/50 dilution; COS celllysat transfected with subSCR2 at 1/10 and 1/50 dilution; COS celllysate transfected wit subSCR3 at 1/10 and 1/50 dilution; and COS celllysate transfected with subSCR2+3 at 1/10 and 1/50 dilution. Molecularweight markers are indicated.

FIG. 4. Semi-quantitative radioimmunoassay (see Materials and Methods)of COS ce lysates. An equivalent number of transfected cells was usedfor each sample. The mea number of counts bound (duplicates) withstandard error is shown against dilution facto; The titration curves arefor lysates of mock (▪), wtSCR (□) and subSCR2+3 () transfected cells.

FIG. 5. Western blot analysis of COS cell serum-free supernatant 168hours aft, transfection with CD46 constructs (see Materials andMethods). Lanes contain 40 μl undiluted supernatant. From left to rightlanes contain supernatant from cells transfecte with the cell surfaceconstruct subSCR2+3; the soluble construct sol-wtSCR; blank; tl solubleconstruct sol-subSCR3; and no DNA (mock). Molecular weight markers aindicated.

FIG. 6. Semi-quantitative radioimmunoassay (see Materials and Methods)of COS cell supernatants. An equivalent amount of tissue culturesupernatant was used for each ample. The mean number of counts bound(duplicates) is shown against the dilution factor. The titration curvesare for supernatants of mock (▪), sol-wtSCR (□) and sol-subSCR3 ()transfected cells.

FIG. 7. Northern blot analysis of CD46 mRNA in transfected COS cells.Total RNA prepared from COS cells 48 hours after transfection wasseparated by electrophoresis (10 μg/track). Northern blotting was asdescribed in Materials and Methods. The position of the 28S and 18Sribosomal RNA bands are indicated. Lanes 1-5 contain RNA prepared fromCOS cells transfected with no DNA, wtSCR, subSCR2, subSCR3 andsubSCR2+3, respectively.

FIG. 8. Southern blot analysis of CD46 mRNA in transfected COS cells.DNA prepared from COS cells 48 hours after transfection was separated byelectrophoresis (5 μg/track). Southern blotting was as described inMaterials and Methods. Lanes 1-5 contain DNA prepared from COS cellstransfected with no DNA, wtSCR, subSCR2, subSCR3 and subSCR2+3,respectively.

FIG. 9A-9D. Flow cytometry of CHO cells after stable transfection withwildtype and mutant constructs. Flow cytometry was performed asdescribed in Materials and Methods. All cells were stained with CD46 mAbE4.3. Panel A shows pgk-neo transfected CHO cells. Panel B showsprofiles for wtSCR transfected cells (-----) overlayed with subSCR2transfected cells (----); panel C shows wtSCR (---) overlayed withsubSCR3 (----); panel D shows wtSCR (---) overlayed with subSCR2+3(----).

FIG. 10. In vitro translation of synthetic CD46 transcripts. Lanescontain 35S-methionine labelled in vitro translation products fromcapped mRNAs as labelled. The arrow indicates the non glycosylatedprotein of the expected size, and the molecular weight markers areshown.

FIG. 11. Semi-quantitative radioimmunoassay (see Materials and Methods)of CHO-K1 cell lysates. An equivalent number of transfected cells wasused for each sample. The mean number of counts bound (duplicates) withstandard error is shown against dilution factor. The titration curvesare for lysates of mock (▪), soluble wtSCR (□) and soluble subSCR3 ()transfected cells.

    ______________________________________                                        Summary of Sequence listings                                                  ______________________________________                                        Seq ID No.1                                                                           The cDNA sequence of wtSCR showing amino acid                                 translation                                                           Seq ID No.2                                                                           The cDNA sequence of subSCR2 showing amino acid                               translation                                                           Seq ID No.3                                                                           The cDNA sequence of subSCR3 showing amino acid                               translation                                                           Seq ID No.4                                                                           The cDNA sequence of subSCR2 + 3 showing amino acid                           translation                                                           Seq ID No.5                                                                           The cDNA sequence of delSCR2subSCR3 showing amino                             acid translation                                                      ______________________________________                                    

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In a first aspect the present invention provides a method of increasingor improving protein production in a host cell where a nucleic acidencoding the protein has an A and/or T rich region present in an exon,said method comprising altering the nucleic acid by reducing or loweringthe amount of A and/or T in said region, transfecting a host cell withsaid nucleic acid and, under appropriate conditions, obtainingexpression of said nucleic acid.

The phrase "increasing or improving protein production" refers tocausing higher production of the desired protein than would routinely beexpected in an host cell and includes enhancing protein production. Themethod of the present invention is particularly directed to providinggreater protein production levels where production levels are less thanoptimal but is not limited to this situation.

The term "host cell" means any cell capable of expressing the nucleicacid encoding the protein. Both prokaryotic and eukaryotic host cellsare contemplated.

The term "nucleic acid encoding a protein" refers to a nucleic acidcomprising DNA or RNA and includes genes containing introns and exonsand cDNA molecules. The term also includes nucleic acids comprising orcontaining synthetic purine and pyrimidine bases.

The phrase "A and/or T rich region" denotes a stretch of nucleotideswhich ontains a higher than average amount of A and/or T, and mayalternatively be referred to as an "AT rich region". This means astretch of nucleotides with a greater than average AT content for theparticular species from which the nucleic acid is derived. For instancean AT rich region in a nucleic acid encoding a human protein would be aregion that contains more than 60% A and/or T in a particular span ofnucleotide sequence. It should be understood that the term AT richregion includes an AU rich region (where RNA is the nucleic acid) andthat "A and/or T" also means "A and/or U" in an RNA context.

The term "exon" means that part of a nucleic acid actually encoding theprotein.

The phrase "altering the nucleic acid by reducing or lowering the amountof A and/or T, in said region" means that A and/or T nucleotides in theAT rich region are replaced by substitution of other bases (such as G orC, or synthetic nucleotides) or that these nucleotides are deleted andnot replaced by another base. Usually such a deletion will be an "inframe" deletion thus not producing a frame-shift. The reduction orlowering the amount of A and/or T includes reference to lowering theproportion of A and/or T with respect to G and/or C nucleotides. Theterm "mutation" is also used to refer to such alterations throughout thespecification.

Where the nucleotides are replaced by other bases the resultantalteration may be silent so that the codon encodes the same amino acid.Silent mutations are well known to those skilled in the art and mostcommonly involve changing the third nucleotide of the codon.

Alternatively, the replacement may be conservative, i.e. results in acodon specific for an amino acid with similar properties to the originalamino acid specified. Suitable conservative amino acid substitutions aregiven in Table A below. Those skilled in the art will know which basesto substitute to confer a conservative amino acid substitution

Typical substitutions are those made in accordance with Table A.

                  TABLE A                                                         ______________________________________                                        Suitable residues for amino acid substitutions                                Original Residue  Exemplary Substitutions                                     ______________________________________                                        Ala               Ser                                                         Arg               Lys                                                         Asn               Gln; His                                                    Asp               Glu                                                         Cys               Ser                                                         Gln               Asn                                                         Glu               Ala                                                         Gly               Pro                                                         His               Asn; Gln                                                    Ile               Leu; Val                                                    Leu               Ile; Val                                                    Lys               Arg; Gln; Glu                                               Met               Leu; Ile                                                    Phe               Met; Leu; Tyr                                               Ser               Thr                                                         Thr               Ser                                                         Trp               Tyr                                                         Tyr               Trp; Phe                                                    Val               Ile; Leu                                                    ______________________________________                                    

Alternatively an entirely different codon may be created by insertion ofa nucleotide other than A or T depending on the protein product desired.It will be understood that this will usually be an in frame insertion.

The term "transfecting" means causing the nucleic acid to be taken up bythe cell. The nucleic acid will usually be present as a component of anexpression vector or the like. The resultant transfection may betransient, where the gene is maintained on an episome, or stable. Stabletransfection is achieved where the gene is integrated into the cellgenome and becomes stably heritable. Nucleic acids can also beintroduced into host cells via viral vectors by infection of host cellswith, for example, recombinant vaccinia or adenoviruses.

The method of the invention contemplates increasing production of allproteins encoded by nucleic acids with AT rich regions in their exonswhere it is desired to produce the proteins in an eukaryotic system. Thenucleic acids encoding candidate proteins may be screened for AT richregions in their exons by standard techniques such as searching theGenBank data base. These nucleic acids include those encoding cellsurface proteins, peptide hormones, cytokines, growth factors, receptorsand their ligands, novel recombinant proteins for therapeutic uses orreagents where it is desired to increase production of the protein.Complement regulating proteins, particularly those containing shortconsensus repeat domains, such as CD46, are of particular interest.

The prokaryotic cell used in the method may by any prokaryotic cellsuitable for expressing the required gene. This includes Escherichiacoli (for example E. coli MC1061, E. coli DH5.sub.α, E. coli NM522, EK12and E. coli W3110), bacilli such as Bacillus subtilis or otherenterobacteriaciae such as, for example, Salmonella typhimurium orSerratia marcesans and various Pseudomonas species. Preferablynon-pathogenic, attenuated strains of these organisms are used.

The eukaryotic cell used in the method may be any eukaryotic cellsuitable for expression of the required gene. Suitable cells which willbe known by those skilled in the art, include animal cells such asCOS-7, CHO-K1, WOP-3027, C127, VERO, HeLa, W138, BHK, 293, MDCK andL929. Others such as Saccharomyces, Kluyveromyces, Pichia pastoris,Schwanniomyces and Hansenula may also be suitable.

Suitable vectors used to transfect the host cell with the nucleic acidin the method will be well known to those skilled in the art. Suitableprokaryotic vectors include pGEX, pKC30, pKK173-3, pET-3 and others.These vectors will preferably contain replicon and control sequencesthat are compatible with the chosen host cell described above. Forexample, in mammalian cells the control functions on eukaryoticexpression vectors are usually provided by viral sources such asAdenovirus 2, polyoma, HIV-1, human cytomegalovirus and SV40.

The phrase "under appropriate conditions" means conditions suitable toobtain expression of the nucleic acid and production of the protein.Culture conditions such as nutrients, temperature and so on will be wellknown to those skilled in the art.

There appears to be a number of possible mechanistic explanations forthe increased level of protein production provided by lowering the Aand/or T content of the AT rich regions present in an exon. Examplesinclude improved stability of transcripts, increased delivery of mRNA tothe cytoplasm, increased efficiency of translation by ribosomes,increased translation due to mRNA superstructure or in fact acombination of two or more of these factors. Without wishing to be boundby theory it appears likely that the increased protein production can beexplained by an increased rate of protein translation. Data indicatingthat the increased protein production is limited to intact cells and isnot evidenced in cell-free translation systems tends to indicate theeffect is due to some interaction between mRNA transcripts, ribosomes,polysomes, RNA-binding proteins and/or other factors such assequence-dependent 3-dimensional mRNA structure rather than substrateavailability (such as for example RNA stability effects) or simplestructural effects or simple protein translation component interaction.

While there is still some speculation about the exact mechanismresponsible for the improved protein production associated with thepresent invention, it is nontheless noted that protein productionincreases of up to or exceeding 20-fold in terms of cell-surfacemolecule expression can be obtained. The increased protein productionappears to be most pronounced, especially in the case of CD46translation, when the reduction in A and/or T content is effected withina region up to about 540 bases from the initiation codon.

In a preferred embodiment the host cell used in the method is aeukaryotic cell. This is because it is often desirable to obtainpost-translational modifications of the recombinant protein, such asglycosylation which cannot always be provided by prokaryotic hosts andsome insect cells.

In another preferred embodiment the nucleic acid encodes an RCA whichhas an A and/or T rich region in one or more of its exons. The RCA maybe CD46, CD55, CD35 or factor H amongst others.

In a particularly preferred embodiment the present invention relates toa method of increasing or improving CD46 production in a eukaryotic cellwhere the gene encoding CD46 is altered by reducing or lowering theamount of A and/or T in an A and T rich region in one or more exons ofsaid gene and transfecting a eukaryotic cell with said altered gene and,under appropriate conditions, obtaining expression of said gene.

The term "CD46" used herein refers to all native isoforms andrecombinant splice variants, including native forms not commonlyexpressed by human cells.

Any CD46 gene natural or recombinant may be used as a starting point forsynthesis of the altered CD46 gene. This includes the native CD46 genesand the CD46 constructs disclosed in PCT/AU91/00199 (the specificationof which is herein incorporated by reference). cDNA CD46 constructswhich by definition comprise exons are preferred. In the Examples hereinclone pm5.1 of PCT/AU91/00199 is used as a starting point.

In a particularly preferred aspect of the invention the alterations aremade to AT rich regions encoding the short consensus repeat (SCR)domains or to putative polyadenylation signals. This is particularly sowhen putative polyadenylation signals occur inappropriately withinprotein-coding exons. More particularly preferred are alterations to theboundary or junction, surrounding the 3rd and 4th exons in CD46 cDNAconstructs. The 3rd and 4th exons encode one of the SCRs and contains astretch of 49 nucleotides 78% which are A or T which may be altered. Inaddition, alterations to a smaller A rich region within the adjacentexon 5 may also be carried out separately or in conjunction with theabove alterations.

Although exons 3, 4 and 5 have been the target for the alterations sofar described other regions of the CD46 gene may be contemplated such asexons 6 and 14.

The alteration may be made by replacing the A or T nucleotides withother bases or deleting the A or T nucleotides and not replacing themwith other bases as described above. The substitutions will depend onthe structure of the protein required and may be silent or conservative.

Still more preferably the AT content of the region between nucleotides406 to 454 of FIG. 1 is reduced from 78% to approximately 60%, morepreferably approximately 55%.

Alternatively, still more preferably the A rich region in SCR3 isreduced, which interrupts a putative poly A signal on the wildtype CD46gene, and the nucleotides substituted extend 5' to the end of the regionencoding SCR2.

Alternatively more preferably the SCR2 is deleted and optionally thereare substitutions in the SCR3.

In addition to the above alteration mutations can also be made whichrender the CD46 protein soluble. Preferably a stop codon is created forexample at position 1022 of pm5.1 (Purcell et al (1991)) by changing anA to a G.

In a still more preferred form the invention relates to a method ofusing the constructs of subSCR2, subSCR3, subSCR2/3 and delSCR2/subSCR3as illustrated in FIG. 1. By way of explanation a construct containingsubstitutions in exons 3 and 4 are denoted subSCR2, substitutions inexon 5 is denoted subSCR3 and substitutions in exons 3, 4 and 5 aredenoted subSCR2+3. The construct which comprises deletion of SCR2 andcontains a substitution in SCR3 is designated delSCR2/subSCR3.

Where substitution of A or T nucleotides is desired splice overlapextension (SOE-PCR) may be used. Other methods such as PCR basedmutagenesis, and ligation of an appropriately mutated syntheticnucleotide will be known to persons skilled in the art. Deletion of A orT nucleotides or stretches of nucleotides may be carried out by standardsite directed mutagenic techniques.

Preferably the initial constructs containing the altered CD46 cDNA aremade in bacterial vectors. Once it has been confirmed that the desiredconstruct has been produced then the altered CD46 gene may be ligatedinto a eukaryotic vector such as pcDNA1, pCDM8, PGK-neo, pEE6.hCMV.GS,pHIV, pHMG or pMJ601 and others.

The desired construct containing the altered gene is then used totransfect an appropriate host cell. Preferably this is a eukaryotic hostcell. Standard transfection techniques may be employed preferably usingLipofectamine (Gibco-BRL, Gaithersburg, USA) or a similar reagent. COS-7or CHO-K1 cells are preferably transfected although other eukaryoticcells may be utilised such as WPO-3027, L929 and so on.

In a related aspect the present invention provides a method forenhancing production of RCA proteins in a host cell where a nucleic acidencoding an RCA has A and/or T rich regions present in one or more ofits SCR modules, said method comprising altering the nucleic acid bylowering the amount of A and/or T in said A and/or T rich regions,transfecting a host cell with said nucleic acid and, under appropriatecondition obtaining expression of said nucleic acid.

The terms "A and/or T rich region" and "lowering the amount of A and/orT" have the same meaning as given above.

Preferably a eukaryotic host cell is used in the method such as thosedescribed above.

More preferably the AT rich regions in SCR2 and SCR3 are subject toalteration.

The above method is based on the observation that alterations innucleotides encoding the SCR2 and SCR3 modules in CD46 produce anadditive effect which greatly increases the amount of protein produced.

Preferably the nucleic acid encodes CD46.

Preferably the alteration is obtained through silent nucleotidesubstitutions.

More preferably the nucleic acid is the construct subSCR2+3 describedherein.

In a second aspect the present invention relates to a method ofincreasing production of an RCA protein in a host cell comprisingaltering a nucleic acid encoding the RCA by deleting one or more SCRmodules with contains an AT rich region, or a part thereof, transfectinga host cell with said altered nucleic acid and under appropriateconditions obtaining expression of said nucleic acid.

Preferably a eukaryotic host cell, such as those described above isemployed in the method.

Optionally, in the above method other parts of the nucleic acid,including AT rich regions outside of the SCR module(s) may also bechanged such as by reducing the amount of A and/or T in the AT richregion.

Preferably the altered nucleic acid encodes a CD46 isoform. Morepreferably the nucleic acid encoding CD46 is delSCR2/subSCR3 describedherein.

In a third aspect the present invention also relates to a recombinantnucleic acid construct which is capable of increased production of aprotein in a host cell wherein the amount of A and/or T in one or more Aand/or T rich regions present in one or more of the exons has beenreduced.

The term "recombinant nucleic acid construct" refers to a molecule madeup of nucleic acids which have been combined from different sources andis capable of transfecting a host cell.

The term "increased production" refers to expression levels higher thanthat demonstrated in previous constructs including those containingnative genes encoding the protein of interest.

The terms "A and/or T rich region", "exon", "host cell" and reduction ofthe amount of the A and/or T in the A and/or T rich region have the samemeanings as given earlier.

Such constructs may be produced by the methods given earlier.

Preferably the host cell is a eukaryotic host cell.

Preferably the constructs comprise nucleic acids which encode proteins,the genes of which in their native state contain A and/or T rich regionsin their exons. The nucleic acids may encode proteins such as cellsurface proteins, peptide hormones, growth factors, receptors and theirligand, novel recombinant proteins for therapeutic uses or reagents.Complement regulating proteins, particularly those containing shortconsensus repeat domains are included.

In a particularly preferred aspect of the invention the constructsencode an altered CD46 cDNA sequence. While any CD46 cDNA may be used asa starting point those disclosed in PCT/AU91/00199 (which isincorporated herein by reference), particularly clone pm5.1 arepreferred.

In a more preferred aspect the construct comprises alterations in one ormore of the following AT rich regions of FIG. 1 of the CD46 construct,or its equivalent nucleotides 406 to 454, 504 to 516 and 530 to 561.

In a preferred aspect of the invention the CD46 constructs of theinvention have been altered so that the proportion or amount of A and/orT in the AT rich regions of the SCR domains has been reduced andoptionally the putative poly A signal have been removed.

In a more preferred aspect of the present invention the constructcomprises alterations which have reduced the amount of A and/or T in theAT rich region of the boundary or junction surrounding the 3rd and 4thexons.

Still more preferred is a construction comprising a reduction of Aand/or T in the 49 nucleotide stretch 78% of which comprises A and/or Tin the 3rd and 4th exons or a reduction in the A rich region within theadjacent exon 5.

In a most preferred aspect the invention relates to the constructssubSCR2, subSCR3, subSCR2+3 and delSCR2/subSCR3.

The above constructs or their derivatives which result in production ofCD46 at the cell surface may be used to produce transgenic animals suchas transgenic pigs whose organs produce cell surface CD46. Such organsmay be used in transplantation therapy. The term "derivative" abovemeans recombinant constructs containing the altered CD46 gene or genesegments derived from the above constructs.

The present invention also relates to a purified preparation of aprotein produced by the first and second aspects of the invention and toa purified preparation of a protein encoded by the constructs of thethird aspect of the invention.

The term "purified preparation" refers to preparation of proteinsseparated from, in at least some degree, the proteins and otherconstituents of the host cell. Preferably the protein is at least 50%pure, preferably 60% pure, more preferably 70% pure, still morepreferably 80% pure and still more preferably 90% pure, or more, whencompared to other proteins or constituents as determined by weight,activity, amino acid similarly antibody reactivity or other convenientmeans.

Preferably the protein is CD46BC1. Most preferably the invention relatesto a soluble CD46 (sCD46(BC)) protein encoded by sol-subSCR3 (seeTable 1) as described or an equivalent.

The invention also relates to host cells expressing the desired proteinmade in accordance with the first and second aspects of the invention ortransformed with the constructs of the third aspect of the invention.

In a particularly preferred aspect the invention relates to stablytransfected host cells or transgenic animals expressing the desiredprotein.

The invention also relates to a method of producing an altered geneencoding a protein wherein said altered gene is capable of causingincreased production of the protein said method comprising altering agene with one or more A and/or T rich regions present in one or moreexons by reducing or lowering the amount to A or T in said regions.

The terms, phrases and methods for producing the altered gene used abovehave been explained earlier.

Preferably the altered gene is capable of causing increased productionof a protein in a eukaryotic host cell.

Preferably the altered gene comprises a CD46 encoding segment.

Preferably the primers described in Table 1 are used in splice overlapextension PCR to produce the altered CD46 gene.

In another aspect the present invention comprises a pharmaceuticalcomposition comprising as an active ingredient a purified proteinpreparation described above together with a pharmaceutically appropriatecarrier or diluent.

The pharmaceutical composition may be used in the treatment of diseasesfor which the active ingredient is indicated. In the case where theactive ingredient is CD46 then the composition may be used to preventcomplement mediated, or inflammation mediated tissue damage, to enhanceimmunity to tumours and viruses, to control the process offertilization, to prevent recurrent spontaneous abortion of the foetusduring pregnancy and to facilitate engrafling of transplanted tissue aspreviously described in PCT/AU91/00199 which is incorporated herein byreference.

The formation of pharmaceutical compositions is generally known in theart and reference can conveniently be made to Remington's PharmaceuticalSciences, 17th ed, Mack Publishing Co, Pennsylvania, USA.

In a preferred aspect the present invention comprises a pharmaceuticalcomposition comprising CD46 made according to the above methods orderivable from the above constructs together with a pharmaceuticallyappropriate carrier or diluent.

In a particularly preferred aspect the present invention comprises apharmaceutical composition comprising the soluble CD46 protein as hereindescribed.

The invention will be further described with reference to the followingnon-limiting examples.

EXAMPLE 1 Materials and Methods

The CD46 Isoform Nomenclature

The CD46 gene produces 4 commonly expressed mRNAs (up to 14 in total) byalternative RNA splicing (Russell et al., 1992). To simplifyrepresentation of CD46 protein isoforms only the variably splicedregions, the STP-A,B,C segments (encoded by exons 7, 8 and 9) andcytoplasmic tails (tail 1 is encoded by exon 13, whereas tail 2 isencoded by exon 14 after the splicing out of exon 13) are denoted unlessmutated for the purposes of the present application. The term "mutated"used here means the same as the alterations discussed earlier. Thewildtype protein isoform produced in this application includes STPs Band C (exons 8, 9) and cytoplasmic tail 1 (exon 13) and is denoted byCD46(BC1)(the equivalent protein is encoded by pm5.1 cDNA in Purcell etal, 1991 and PCT/AU91/00199). The protein isoform containing STPs B andC and cytoplasmic tail 2 is denoted by CD46(BC2). The soluble proteinproduced here is referred to as sCD46(BC), because the stop codon is atthe predicted transmembrane boundary.

Nucleotide substituted constructs and products are designated by theprotein domain encoded by the targeted exons. Thus the constructcontaining substitutions in exons 3 and 4 is subSCR2; substitution toexon 5 is subSCR3; and substitutions to exons 3, 4 and 5 is subSCR2+3.The construct which encodes an SCR2 deletion and contains SCR3substitutions is designated delSCR2/subSCR3.

Mutagenesis of CD46 cDNA

Silent nucleotide substitution was achieved using Splice OverlapExtension PCR (SOE-PCR) (Horton et al., 1988). Firstly, two PCRreactions amplified two overlapping DNA fragments which contained therequired mutations in complementary sequences at one end and EcoRI sitesat the other end. Secondly, the two fragments were spliced together in aSOE-PCR reaction using the primers containing the EcoRI sites. The PCRprimers and the nucleotide positions to which they annealed are shown inTable 1A. The overlapping DNA fragments containing mutations wereproduced in two PCR reactions using the templates and oligonucleotidecombinations shown in Table 1B.

The products of the first two PCR reactions for each mutant, the 5' and3' PCR fragments, were isolated after electrophoresis in low-meltagarose (Nusieve), diluted and used directly as template for the SOE-PCRreactions. All the SOE-PCR reactions, including the unrutated wildtypeCD46, used On232 and On241 as oligonucleotides. The PCR and SOEreactions were performed in a DNA Thermal Cycler (Perkin Elmer Cetus)using Amplitaq enzyme and buffer (Perkin Elmer Cetus). The cycleconditions for the first two PCR were 3 cycles of 2 min at 94° C., 1 minat 50° C. and 1 min at 72° C. followed by 22 cycles of 30 sec at 94° C.,1 min at 60° C. and 1 min at 72° C. The SOE-PCR cycle conditions were 3cycles of 2 min at 94° C., 1 min at 44° C. and 2 min at 72° C., followedby 22 cycles of 30 sec at 94° C., 1 min at 60° C. and 2 min at 72° C.

While not wishing to be bound by theory the above mutations appear torestore the usual AT/GC balance (proportion of AT:GC) found inmarnmalian nucleic acids and this restoration of balance appears toresult in increased protein production.

Mutant DNA Constructs and Expression Vectors

PCR products were end-filled with DNA polymerase (Klenow fragment)(Boehringer Mannheim) and phosphorylated with T4 polynucleotide kinase(New England Biolabs). The DNA was purified after agarose gelelectrophoresis using NA45 paper (DEAE)(Schleicher and Schuell) followedby MagicPrep DNA clean-up system (Promega Corp.). The DNA products wereligated (DNA ligase, New England Biolabs) into the Sma I site ofpBluescript II SK (Stratagene) and transformed into XL1 Blue E. coli(Stratagene). Plasmid DNA was prepared of clones containing mutationsusing Maxiprep (Promega Corp.) and the entire sequence was confirmed bydideoxy sequencing using a Sequenase™ kit (United States BiochemicalCorp.). All products contained the nucleotide mutations without PCRerror. The DNA inserts were released from the pBluescript vector bydigestion with EcoRI, end-filled with DNA polymerase (Klenow fragment)(Boehringer Mannheim), ligated into the Eco RV site of pcDNA I(Invitrogen Corp.) and transformed into MC1061/p3 E. coli. The cloningsites were confirmed by dideoxy sequencing. Plasmid DNA for transfectionwas prepared using Wizard™ Maxiprep DNA purification system (PromegaCorp).

Transfection

COS-7 green monkey fibroblasts and CHO-K1 Chinese hamster ovaryfibroblasts were cultured in DMEM medium supplemented with 20 μg/mlglutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% v/vfoetal bovine serum (CHO-K1 medium also contained 20 μg/ml proline, 14μg/ml hypoxanthine and 3.9 μg/ml thymidine), in a humidified 10% CO₂atmosphere.

Plasmid DNA (pcDNA1-CD46 constructs) was introduced into cultured celllines with Lipofectamine (Gibco-BRL) in serum-free and antibiotic-freeDMEM medium, according to the manufacturer's instructions. One six-wellplate of COS-7 cells was transfected with each construct. The cellstransfected with cell-surface CD46(BC1) were harvested 48 hours aftertransfection and were split into aliquots to be analysed by Southern,Northern and Western blotting, radioimmunoassay (RIA) of cell lysates,fluorescent microscopy and flow cytometry. For the expression of solubleCD46 constructs, the COS-7 cells were cultured in serum-free mediumwhich was collected 120 or 148 hours after transfection. For generationof transfected CHO-K1 cells with stable construct integration, PGK-neoplasmid (a gift from Dr Richard Harvey, WEHI, Melbourne, Australia) wascotransfected, with a 10-fold excess molar concentration of thepcDNA1-CD46 construct plasmid DNA. Transfected CHO cells were selectedfor stable expressors with 1.2 mg/ml G418 and assayed for cell surfaceexpression of CD46 after two or more weeks.

Analysis of Cell Surface CD46(BC1) Production by Immunofluorescence

CD46 protein processed on the plasma membrane, or as intracellularprotein in permeabilised cells, was assayed by two-stageimmunofluoresence labelling. The primary anti-CD46 mAb (E4.3 IgG2a,Sparrow & McKenzie 1983; or M177 IgGI, Seya et al, 1990) or isotypecontrol mAb (MEM-43 IgG2a anti-CD59, Stefanova et al, 1989) wasincubated with cells for more than 30 min on ice, cells were washed andFITC sheep(Fab)-anti-mouse Ig (Silenus, Melbourne, Australia) was addedfor a further 30 min on ice. Fluorescence was assessed by flow cytometry(FACScan II, Becton Dickinson) of cell suspensions or by microscopy ofnear-confluent cell monolayers.

Analyses of CD46(BC1) in Whole Cell Lysates

Whole cell lysates were prepared as described at a concentration of5×10⁶ cells/ml. An equivalent number of cells was used for all samplesand Western blotting was performed as described (Laemmli, 1970). Theproteins present in the lysate were separated by non-reducing SDS-PAGE,blotted and probed with the mAb E4.3. The capture/tracer RIA used mAbE4.3 to capture the CD46 protein and ¹²⁵ I-labelled M177 (a gift fromSeya) as the tracer and was performed as described, but withapproximately 10000 cpm input counts of the tracer (Johnstone et al.,1993).

Analyses of DNA and RNA

RNA was prepared using a modification of the procedure of Chirgwin etal. (1979). Northern blotting was performed as described (Milland etal., 1990) except that the membrane was Hybond-N (Amersham) and thetransfer was by gravity in a downwards direction. Cellular DNA wasprepared and Southern blotting was performec as described (Southern)using Hybond-N⁺ (Amersham) as the membrane. For the Northerns andSoutherns the probe used for hybridisation was pm5.1 cDNA (Purcell etal, 1991), labelled using the Megaprime DNA Labelling System (Amersham).

Plasmid Constructs

The sequences of the SCR mutation are shown in FIG. 1. All the finalconstructs were in the pcDNA1 vector and had the same cloning sitesderived from the oligonucleotides used for the PCR. The constructcontaining unrmutated (wtSCR) sequence is derived from the originalpm5.1 cDNA clone (Purcell et al., 1991). This and each of the silentsubstitution constructs discussed below encodes wildtype CD46(BC1)protein. The subSCR2 construct contains silent nucleotide substitutionsin the region in SCR2 around the exon 3/4 splice site and encodeswildtype CD46(BC1) protein. The substitutions reduce the A/T content ofthe region between the nucleotides 406 and 454 from 78% to 55%. ThesubSCR3 construct contains silent nucleotide substitutions to an A-richregion in SCR3 (including one substitution at the end of SCR2) and alsointerrupts a putative polyadenylation signal and encodes wildtypeCD46(BC1) protein. The subSCR2+3 construct contains a combination of theSCR2 and SCR3 changes and encodes wildtype CD46(BC1) protein. The finalcell surface construct, delSCR2/subSCR3, has SCR2 deleted andsubstitutions to SCR3. This construct encodes a mutant protein withoutSCR2, called CD46(delSCR2/BC1). Two DNA constructs were mutated toencode a soluble protein called sCD46(BC). The first construct,sol-wtSCR was produced by the substitution of an A for a G at nucleotide1022 to produce a stop codon before the transmembrane domain. Thesol-subSCR3 construct contains the stop codon mutation and the silentnucleotide substitutions in SCR3 described above.

Silent Nucleotide Substitution Increases the Production of Cell SurfaceCD46(BC1)

Initially the inventors showed that mutation increased proteinproduction CD46 by photomicrography. In these experiments cells weretransfected in wells and stained with E4.3. Results are not shown. Theorder of increasing protein production was wtSCR, subSCR2, subSCR3,subSCR2+3 and delSCR2/subSCR3. The improvement in CD46 production wasalso observed after transfection of WOP-3027 cells (data not shown).

Flow cytometry showed that more COS-7 cells were producing CD46 on theirsurface and that the cells were also producing at a significantly higherlevel after transfection with the mutated constructs compared to thewtSCR construct (FIG. 2). The improvement in production was in the sameorder as for the photomicrography. As would be expected, a proportion ofthe cell population has a similar fluorescence to the mock transfectedcells and represent untransfected or transfected cells without cellsurface CD46, however, in panels C, D and E, it appears that all cellsexposed to the mutated constructs were transfected and expressed CD46:the entire profile in each case is shifted to the right in comparison tothe profile of wildtype transfected cells.

Silent Nucleotide Substitution Increases the Production of CD46(BC1) inCell Lysates

Cell lysates were prepared from transfected cells. These lysates includeall cel. that were transfected including the large proportion of cellsthat do not produce significant amounts of cell surface CD46. In allcases, the protein produced after transfection of CD46(BC1) was theexpected size (66 kDa) by Western blotting (FIG. 3). There is more CD46in the lysates from cells transfected with mutated CD46 DNA comparedwith unmutated CD46 DNA. The order of improvement in production was thesame as for cell surface expression and cells transfected with thesubSCR2+3 construct gave the best result compared to the wtSCRconstruct.

To enable a better estimate of the difference in the level of CD46proteins, RIA was performed (FIG. 4). The population of cellstransfected with subSCR2 or subSCR3 had approximately twice as much CD46in their lysates compared to the wtSCR transfected cells. The cellstransfected with subSCR2+3 had approximately eight times as much CD46 intheir lysates compared to the wtSCR transfected cells. These resultssuggest that the nucleotide sequences in SCR2 and SCR3 may beinteracting in some way, since the silent substitutions to SCR2 and SCR3appear to improve the level of protein expression in an additive way.There may also be other nucleotide sequences in SCR2 affecting the levelof protein expression, since the total deletion of SCR2 increases thelevel of expression more than the silent nucleotide substitutions ofSCR2+3.

Silent Nucleotide Substitution Increases the Production of SolubleCD46(BC)

The silent nucleotide substitutions in SCR3 also enabled significantlygreater quantities of sCD46(BC) to be expressed in a transient COSsystem. The sol-subSCR3 construct produced protein detectable by Westernblotting in the serum-free supernatant of cells 148 hours aftertransfection of COS-7 cells (FIG. 5). Supernatant from similarlytransfected cells 120 hours post-transfection was analysed by RIA. Thelevel of soluble material secreted by cells transfected with sol-subSCR3was approximately nine times that of cells transfected with sol-wtSCR(FIG. 6). COS cells transfected with the subSCR2+3 (shown in FIG. 5) ordelSCR2/subSCR3 (data not shown) also shed or secreted significantamounts of CD46 protein into the tissue culture supernatant which was inaddition to CD46 expressed on the cell surface. None of the other cellsurface transfectants produced detectable quantities of CD46 in thesupernatant.

The Increase in Production of CD46(BC1) Caused by Silent NucleotideSubstitutions does not Appear to be Due to Increases in Expression ofmRNA or of Transfected DNA

Total RNA was prepared from populations of cells transfected with thevarious constructs and level of mRNA expression was determined byNorthern blot (FIG. 7). The level of mRNA in total RNA was similar forconstructs containing the silent mutations compared with the level forthe unmutated wtSCR construct (FIG. 7). This level of expressionrepresents that of the total cell population and does not indicate thelevel of expression per cell. The amount of plasmid present in thepopulation of cells was determined by Southern blotting (FIG. 8) and wasnot significantly different for any of the constructs.

Silent Nucleotide Substitutions Increase the Production of CD46(BC1) ina Stable Transfection System

After transfection and G418 drug selection, CHO-K1 transfectants wereanalysed by flow cytometry. Populations of selected cells transfectedwith constructs containing silent mutations contained more cellsexpressing CD46 and they expressed CD46 to a much a higher level (FIG.9), with the greatest increase observed for the subSCR2+3 construct(panel D).

EXAMPLE 2

Preparation of mutagenised CD55 constructs.

A particular example for application of the invention can be provided bythe nucleic acid encoding CD55 (DAF: Decay Accelerating Factor), astructurally related RCA molecule. The CD55 gene has AT-nucleotide richregions within exons encoding the SCR2, SCR3 and SCR4 modules of CD55.Silent nucleotide substitutions are directed individually toward thesequences included within residues 388 to 412 (encoding SCR2); 590 to602, 633 to 645 and 658 to 674 (encoding SCR3); 718 to 728 and 844 to854 (encoding SCR4). The particular PCR primers for mutagenesis aredesigned to reduce the A and/or T content of the codons for theseregions without altering the predicted amino acid sequence, which wouldbe clear to one skilled in the art, but have sufficient lengths ofsequence complementary to the cDNA template to enable priming of theinitial PCR. Numbering of the CD55 gene is according to FIG. 3 of Lublinand Atkinson (Curr Top Micro Immunol 153, 123, 1989). These primers aredesigned in complementary pairs (forward and reverse primers in 5' and3' directions) to allow annealing of the mutated PCR products in theSplice-Overlap Extension PCR as described specifically above. Additionalforward and reverse PCR primers are designed, containing restrictionenzyme cleavage sites, which anneal to the wildtype cDNA template atpositions flanking the 5'-UTR and 3' of putative polyadenylation signalsin the 3'-UTR, as is obvious to one skilled in the art. The six AT-richsequences identified and listed above may variously accord with therequirements for augmented production of the protein in transfectedcells. Each mutated construct is created individually, and combinationsincluding multiple mutated spans of nucleotide sequence can beadditionally prepared.

The PCR products are purified and cloned into the SK vector for completesequencing to confirm integrity of the required mutated sequence, beforecloning into the appropriate expression vector (e.g., pcDNA1) andtransfection into eukaryotic cells. Production of the CD55 protein isassessed by flow cytometry using the IH4 anti-CD55 monoclonal antibody,and the relative expression of CD55 in transfected cells is correlatedwith the mutagenised construct. Thus the hierarchy of mutationsaffecting protein translation can be constructed.

Cells transfected with the optimal constructs are further tested forprotection against complement-mediated lysis mediated by the expressedCD55, as decay-accelerating activity. This assay proves that the proteinis appropriately expressed and is functioning normally.

EXAMPLE 3

Summary of CD46-Transfection Data

Nucleotide constructs, either of wild-type or mutated (substitution)forms, were cloned into the expression vectors: pcDNA1 (as alreadydescribed); and APEX-3.

These expression constructs were transfected into cultured mammaliancell lines. Transfections were repeated to ensure that any variations inexpression recorded were consistent and not erratic artefacts. ThepcDNA1 vector constructs in COS-7 cells (transient expression) werevariously expressed, depending on the silent nucleotide mutations in theparticular CD46 constructs. This effect was consistent both forconstructs encoding cell surface CD46 and constructs encoding solubleCD46. Other cell lines were tested for expression of wild-type andmutated constructs: mutation improved cell-surface CD46 proteinexpression in transiently transfected WOP-3027 cells, and in stablytransfected CHO-K1 cells. Soluble protein was also produced withconsistent and predictable expression in human 293 and 293-Ebna cells.Therefore, the effect of the mutation is not limited to COS-7 AfricanGreen monkey kidney fibroblast-like cells, but also is seen in WOP-3027(polyoma virus-transformed) mouse embryonal fibroblast cells, CHO-K1hamster ovary cells, and 293 (human embryonal kidney) cells. It istherefore likely that the effects will be observed in other cells also.

To assess the effect of the vector upon the expression of mutatedconstructs, forms encoding soluble CD46 were cloned into the APEX-3vector. Mutation-dependent enhanced expression was seen in COS-7 and 293(human embryonal kidney) cells, demonstrating that the effect was notlimited to one vector.

Table II shows cell surface and soluble CD46 expression levels obtainingby transfection using a range of vectors, cell lines and constructs. The"±" symbol indicates basal, wild type expression levels and the numberof "+" symbols shown for each test is directly proportional to the levelof CD46 expression.

EXAMPLE 4

Studies of Translation Rate in Cell-free Translation Systems.

CD46 synthetic wildtype and mutant transcripts were translated in vitroin a cell-free translation system containing rabbit reticulocytes (seeFIG. 10). The lanes containing equal quantities of RNA transcribed fromwtSCR, subSCR2, subSCR3 and subSCR2+3 DNA sequences all produced asimilar amount of protein, showing that translation proceeded withequivalent efficiency regardless of the sequence of the transcript. Theprotein was of the expected size for nonglycosylated CD46 (38-40 kD).

EXAMPLE 5

Enhanced Expression of Soluble CD46 in CHO-K1, WOP-3027 and 293 Cells.

The soluble wtSCR and soluble subSCR3 constructs were transfected intoWOP, CHO-K1 and 293 cells. An almost identical trend was observed whensupernatants collected from transfected WOP cells were semi-quantitatedby radioimmunoassay. Serum free supernatants from CHO cells collected 96hours after transient transfection with soluble subSCR3, showed a 4-foldincrease in protein expression (see FIG. 11). This was consistent withthe increase observed when a parental stable CHO cell line expressingsurface CD46 was established after cotransfection with soluble subSCR2+3and pgKNeopA. Serum free supernatants from 293 cells were collected 6days after transfection, and semi-quantitated by dot blotting, moreprotein (typically 3-fold) was observed when the construct containingsilent nucleotide changes (soluble subSCR3) was used. These data clearlyshow the increase in soluble CD46 protein produced after transfectionwith a mutated construct, was not restricted to COS-7 cells.

EXAMPLE 6

Enhanced Expression of Soluble CD46 Using APEX-3 Expression Plasmids

Inserts encoding CD46 were ligated into the APEX-3 expression plasmid,and the two constructs; APEX3-sol-wtSCR and APEX3-sol-subSCR3transfected into COS-7 & 293 cells. Serum free supernatants werecollected 6 days after transfection, and semiquantitated by dotblotting. An increase in protein expression (typicall 3-fold) wasobserved when cells were transfected with the construct containingsilent nucleotide changes (APEX3-sol-subSCR3). The data confirm that theobserved increase in protein expression was not plasmid specific.

EXAMPLE 7

Studies to Determine Mechanism of Enhanced Protein Expression.

a) Transiently transfected COS cells contain similar quantities ofplasmid DNA whether transfected with CD46 constructs consisting ofmutated (silent nucleotide substituted) or wildtype sequences, asmeasured by Southern blot analyses. Therefore, the significant increasein production of CD46 protein is most likely due to transcriptional ortranslational regulation, and not due to altered efficiency oftransfection, DNA stability, or plasmid DNA replication.

(b) COS cells transfected with mutated or wildtype CD46 plasmidconstructs have equivalent quantities of CD46 mRNA but differentquantities of CD46 protein. In two constructs (subSCR3 and subSCR2+3), acryptic polyadenylation signal has been removed, which might havegenerated truncated mRNA species (in wildtype, delSCR2 or subSCR2constructs). However, no truncated mRNA species are seen in Northernblots, and no differences in mRNA quantity, which might have beenascribed to altered mRNA stability or half-life, are observed.Therefore, the increased CD46 protein is most likely generated byregulation of translation.

(c) In vitro translation, using as template equivalent amounts of mRNAisolated from each of the transfected COS cells, generates equivalentquantities of CD46 protein whereas up to a 20-fold increase incell-surface protein expression is reproducibly found in transfected COScells. This focuses attention on the mechanism of protein translation,as it appears to be limited to specifically occurring within intactcells.

(d) To determine whether the increase in CD46 production was due toprotein COS-7 cells were separately transfected with wtSCR, subSCR2+3 orsubSCR3, biosynthetically labelled with 35S-Cys and 35S-Met, and CD46protein was immunoprecipitated from cell lysates and supernatants andexamined by SDS-PAGE. Cells transfected with subSCR2+3 synthesised morelabelled CD46 than than those transfected with wtSCR. There was no CD46protein detectable in the supernatant. Biosynthetically labelled CD46was produced by cells transfected with solSCR3 and was secreted into thesupernatant but, as expected, could not be detected in the lysate.Lysates and supernatants immunoprecipitated with an isotype control mAb,MEM-43, did not show any labelled material of the expected size.

EXAMPLE 8

The use of Mutagenised CD46 and CD55 Genes, Encoding Wildtype Protein,for the Production of Transgenic Animals.

Once the optimised mutant construct, such as the CD46 encoding SCR2+3construct (see above) or the appropriate CD55 construct, has been provenby repeated transfection of eukaryotic cell lines in vitro, the DNAvector is highly purified to be suitable for transgenesis. The DNA (thetransgene) is injected into fertilised mouse single-cell oocytes whichare reimplanted into prepared pseudo-pregnant mice, entirely accordingto procedures familiar with those experienced in the art. The offspringare screened for integration of the transgene by Southern blotting DNAobtained from a small tissue sample, and founder mice are tested fortransgene expression by Western blotting of a tissue cell lysate.Founders expressing the protein are bred and appropriately backcrossedto suitable mouse strains. Individual mice of each generation are alsoscreened for protein expression of the transgene, and only expressinganimals are used. Tissue samples from such transgenic mice are testedfor function of the transgenic protein in complement-mediated lysis,complement-mediated graft rejection, and complement-mediated tissueinflammation assays, each of which is established and routine.

Only animals expressing sufficient amounts of the CD46 or CD55transgenic protein on their cells to have augmented resistance tocomplement-mediated cell damage are kept to establish transgenic strainsof mice. are kept to establish transgenic strains of mice.

Once function is confirmed using tissues of transgenic mice, whichidentifies the appropriate transgene DNA construct, the sametransgenesis procedure is repeated using porcine oocytes, to producetransgenic pigs. The appropriate transgenic pigs are considered to beputative organ donors of xenogenic transplants for humans requiringkidney, heart, liver, pancreas, etc., grafts.

An important application of the invention is the genetic cross betweentransgenic animals expressing CD46 with transgenic animals expressingCD55, where co-expression of both molecules augments resistance tocomplement-mediated injury.

Although the above example relates to production of transgenic animalsto provide donors of organ implants, transgenic animals containing theconstructs of the present invention or whose cells produce proteins inaccordance with the method of the invention, may be produced for otherpurposes. For example it may be desired to produce a transgenic cowwhich produces recombinant protein in its milk.

It is to be recognised that the present invention has been described byway of example only and that modifications and/or alterations whichwould be obvious to a person skilled in the art can be made thereto,without departing from the intended scope of the invention as defined inthe appended claims.

                                      TABLE I                                     __________________________________________________________________________    Oligonucleotides and the combinations used to generate mutated CD46           constructs.                                                                   __________________________________________________________________________    A. Oligonucleotide primers for PCR. (SEQ ID Nos: 7-15)                        PRIMER                                                                             SEQUENCE.sup.a                   SIZE                                                                              ANNEALING POSITION.sup.b            __________________________________________________________________________    On231                                                                              5'-CAATCAGGTAGTAACCCTCGTTGCAGATGAAGTGCATC-3'                                                                   38-mer                                                                            nt 436-399                          On232                                                                              5'-cggaattcACAGCGTCTTCCGCGCCGCGC-3'                                                                            29-mer                                                                            nt 4-32                             On233                                                                              5'-GGTCTTGTGTACACCACCTCCAAAGATCAAGAATGGAAAAC-3'                                                                41-mer                                                                            nt 510-550                          On238                                                                              5'-AGCAATGACCTAAACATCCAAACTGTC-3'                                                                              27-mer                                                                            nt 1032-1006                        On239                                                                              5'-TGGATGTTTAGGTCATTGCTGTGATT-3' 26-mer                                                                            nt 1013-1038                        On240                                                                              5'-GGTGTACACAAGACCTTATAACAGGCGTCATCTG-3'                                                                       34-mer                                                                            nt 524-509/319-302                  On241                                                                              5'-acgaattcGATTTCAAGCCACTTTCTTTACAAAG-3'                                                                       34-mer                                                                            nt 1641-1608                        On247                                                                              5'-GGTGGTGTACACAAGACCTTCTCACATATTGG-3'                                                                         32-mer                                                                            nt 527-496                          On250                                                                              5'-CGAGGGTTACTACCTGATTGGTGAGGAGATCCTGTATTGTGAAC-3'                                                             44-mer                                                                            nt 417-460                          __________________________________________________________________________    B. Primer combinations for mutated CD46 constructs produced by PCR.           MUTATED        5' PCR       3' PCR                                            CONSTRUCT      FRAGMENT     FRAGMENT      TEMPLATE                            __________________________________________________________________________    subSCR2        On232/On231  On241/On250   pm5.1                               subSCR3        On232/On247  On241/On233   pm5.1                               subSCR2 + 3    On232/On231  On241/On250   subSCR3                             delSCR2/subSCR3                                                                              On232/On240  On241/On247   pm5.1                               sol-wtSCR      On232/On238  On241/On239   pm5.1                               sol-subSCR3    On232/On247  On241/On233   sol-wtSCR                           __________________________________________________________________________     .sup.a EcoRI restriction sites and overhang are shown in lower case;          mutated nucleotides are in italics and bold font.                             .sup.b Annealing position is based on pm5.1 (Purcell et al., 1991)       

                                      TABLE II                                    __________________________________________________________________________    Cell surface and soluble CD46 expression obtained                             by transfection of constructs into various cell lines                                Cell-surface CD46 Construct                                                                  Soluble-CD46 Construct                                         wild-    sub                                                                              del2                                                                             wild- sub                                                                              del2                                                                             1.8                                                type                                                                             sub2                                                                             sub3                                                                             2 + 3                                                                            sub3                                                                             type                                                                             sub3                                                                             2 + 3                                                                            sub3                                                                             mutant                                      __________________________________________________________________________    pcDNA1 vector                                                                 COS-7  ±                                                                             ++ +++                                                                              ++++                                                                             ++++                                                                             ±                                                                             ++ ++ +++                                                                              +++                                         293                   ±                                                                             +  ++                                                293-Ebna              ±                                                                             +  ++                                                WOP-3027                                                                             ±        +++                                                                              ±                                                                             ++ +                                                 CHO-K1 ±                                                                             +  ++ +++                                                                              +++                                                        APEX-3 vector                                                                 COS-7                 ±                                                                             ++ ++                                                293                   ±                                                                             ++ ++                                                __________________________________________________________________________

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - <160> NUMBER OF SEQ ID NOS: 15                                              - <210> SEQ ID NO 1                                                           <211> LENGTH: 1134                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:  CD46  cDNAN: Description of Artificial                                  sequence with wild type SCR                                             - <400> SEQUENCE: 1                                                           - atggagcctc ccggccgccg cgagtgtccc tttccttcct ggcgctttcc tg - #ggttgctt         60                                                                          - ctggcggcca tggtgttgct gctgtactcc ttctccgatg cctgtgagga gc - #caccaaca        120                                                                          - tttgaagcta tggagctcat tggtaaacca aaaccctact atgagattgg tg - #aacgagta        180                                                                          - gattataagt gtaaaaaagg atacttctat atacctcctc ttgccaccca ta - #ctatttgt        240                                                                          - gatcggaatc atacatggct acctgtctca gatgacgcct gttatagaga aa - #catgtcca        300                                                                          - tatatacggg atcctttaaa tggccaagca gtccctgcaa atgggactta cg - #agtttggt        360                                                                          - tatcagatgc actttatttg taatgagggt tattacttaa ttggtgaaga aa - #ttctatat        420                                                                          - tgtgaactta aaggatcagt agcaatttgg agcggtaagc ccccaatatg tg - #aaaaggtt        480                                                                          - ttgtgtacac cacctccaaa aataaaaaat ggaaaacaca cctttagtga ag - #tagaagta        540                                                                          - tttgagtatc ttgatgcagt aacttatagt tgtgatcctg cacctggacc ag - #atccattt        600                                                                          - tcacttattg gagagagcac gatttattgt ggtgacaatt cagtgtggag tc - #gtgctgct        660                                                                          - ccagagtgta aagtggtcaa atgtcgattt ccagtagtcg aaaatggaaa ac - #agatatca        720                                                                          - ggatttggaa aaaaatttta ctacaaagca acagttatgt ttgaatgcga ta - #agggtttt        780                                                                          - tacctcgatg gcagcgacac aattgtctgt gacagtaaca gtacttggga tc - #ccccagtt        840                                                                          - ccaaagtgtc ttaaagtgtc gacttcttcc actacaaaat ctccagcgtc ca - #gtgcctca        900                                                                          - ggtcctaggc ctacttacaa gcctccagtc tcaaattatc caggatatcc ta - #aacctgag        960                                                                          - gaaggaatac ttgacagttt ggatgtttgg gtcattgctg tgattgttat tg - #ccatagtt       1020                                                                          - gttggagttg cagtaatttg tgttgtcccg tacagatatc ttcaaaggag ga - #agaagaaa       1080                                                                          - ggcacatacc taactgatga gacccacaga gaagtaaaat ttacttctct ct - #ga             1134                                                                          - <210> SEQ ID NO 2                                                           <211> LENGTH: 1134                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:  CD46 cDNAON: Description of Artificial                                  subSCR2 variant                                                         - <400> SEQUENCE: 2                                                           - atggagcctc ccggccgccg cgagtgtccc tttccttcct ggcgctttcc tg - #ggttgctt         60                                                                          - ctggcggcca tggtgttgct gctgtactcc ttctccgatg cctgtgagga gc - #caccaaca        120                                                                          - tttgaagcta tggagctcat tggtaaacca aaaccctact atgagattgg tg - #aacgagta        180                                                                          - gattataagt gtaaaaaagg atacttctat atacctcctc ttgccaccca ta - #ctatttgt        240                                                                          - gatcggaatc atacatggct acctgtctca gatgacgcct gttatagaga aa - #catgtcca        300                                                                          - tatatacggg atcctttaaa tggccaagca gtccctgcaa atgggactta cg - #agtttggt        360                                                                          - tatcagatgc actctacttg caacgagggt tactacctga ttggtgagga ga - #tcctgtat        420                                                                          - tgtgaactta aaggatcagt agcaatttgg agcggtaagc ccccaatatg tg - #aaaaggtt        480                                                                          - ttgtgtacac cacctccaaa aataaaaaat ggaaaacaca cctttagtga ag - #tagaagta        540                                                                          - tttgagtatc ttgatgcagt aacttatagt tgtgatcctg cacctggacc ag - #atccattt        600                                                                          - tcacttattg gagagagcac gatttattgt ggtgacaatt cagtgtggag tc - #gtgctgct        660                                                                          - ccagagtgta aagtggtcaa atgtcgattt ccagtagtcg aaaatggaaa ac - #agatatca        720                                                                          - ggatttggaa aaaaatttta ctacaaagca acagttatgt ttgaatgcga ta - #agggtttt        780                                                                          - tacctcgatg gcagcgacac aattgtctgt gacagtaaca gtacttggga tc - #ccccagtt        840                                                                          - ccaaagtgtc ttaaagtgtc gacttcttcc actacaaaat ctccagcgtc ca - #gtgcctca        900                                                                          - ggtcctaggc ctacttacaa gcctccagtc tcaaattatc caggatatcc ta - #aacctgag        960                                                                          - gaaggaatac ttgacagttt ggatgtttgg gtcattgctg tgattgttat tg - #ccatagtt       1020                                                                          - gttggagttg cagtaatttg tgttgtcccg tacagatatc ttcaaaggag ga - #agaagaaa       1080                                                                          - ggcacatacc taactgatga gacccacaga gaagtaaaat ttacttctct ct - #ga             1134                                                                          - <210> SEQ ID NO 3                                                           <211> LENGTH: 1134                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:  CD46 cDNAON: Description of Artificial                                  subSCR3 variant                                                         - <400> SEQUENCE: 3                                                           - atggagcctc ccggccgccg cgagtgtccc tttccttcct ggcgctttcc tg - #ggttgctt         60                                                                          - ctggcggcca tggtgttgct gctgtactcc ttctccgatg cctgtgagga gc - #caccaaca        120                                                                          - tttgaagcta tggagctcat tggtaaacca aaaccctact atgagattgg tg - #aacgagta        180                                                                          - gattataagt gtaaaaaagg atacttctat atacctcctc ttgccaccca ta - #ctatttgt        240                                                                          - gatcggaatc atacatggct acctgtctca gatgacgcct gttatagaga aa - #catgtcca        300                                                                          - tatatacggg atcctttaaa tggccaagca gtccctgcaa atgggactta cg - #agtttggt        360                                                                          - tatcagatgc actttatttg taatgagggt tattacttaa ttggtgaaga aa - #ttctatat        420                                                                          - tgtgaactta aaggatcagt agcaatttgg agcggtaagc ccccaatatg tg - #aaaaggtt        480                                                                          - ttgtgtacac cacctccaaa gatcaagaat ggaaaacaca cctttagtga ag - #tagaagta        540                                                                          - tttgagtatc ttgatgcagt aacttatagt tgtgatcctg cacctggacc ag - #atccattt        600                                                                          - tcacttattg gagagagcac gatttattgt ggtgacaatt cagtgtggag tc - #gtgctgct        660                                                                          - ccagagtgta aagtggtcaa atgtcgattt ccagtagtcg aaaatggaaa ac - #agatatca        720                                                                          - ggatttggaa aaaaatttta ctacaaagca acagttatgt ttgaatgcga ta - #agggtttt        780                                                                          - tacctcgatg gcagcgacac aattgtctgt gacagtaaca gtacttggga tc - #ccccagtt        840                                                                          - ccaaagtgtc ttaaagtgtc gacttcttcc actacaaaat ctccagcgtc ca - #gtgcctca        900                                                                          - ggtcctaggc ctacttacaa gcctccagtc tcaaattatc caggatatcc ta - #aacctgag        960                                                                          - gaaggaatac ttgacagttt ggatgtttgg gtcattgctg tgattgttat tg - #ccatagtt       1020                                                                          - gttggagttg cagtaatttg tgttgtcccg tacagatatc ttcaaaggag ga - #agaagaaa       1080                                                                          - ggcacatacc taactgatga gacccacaga gaagtaaaat ttacttctct ct - #ga             1134                                                                          - <210> SEQ ID NO 4                                                           <211> LENGTH: 1134                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:  CD46 cDNAON: Description of Artificial                                  subSCR2+3 variant                                                       - <400> SEQUENCE: 4                                                           - atggagcctc ccggccgccg cgagtgtccc tttccttcct ggcgctttcc tg - #ggttgctt         60                                                                          - ctggcggcca tggtgttgct gctgtactcc ttctccgatg cctgtgagga gc - #caccaaca        120                                                                          - tttgaagcta tggagctcat tggtaaacca aaaccctact atgagattgg tg - #aacgagta        180                                                                          - gattataagt gtaaaaaagg atacttctat atacctcctc ttgccaccca ta - #ctatttgt        240                                                                          - gatcggaatc atacatggct acctgtctca gatgacgcct gttatagaga aa - #catgtcca        300                                                                          - tatatacggg atcctttaaa tggccaagca gtccctgcaa atgggactta cg - #agtttggt        360                                                                          - tatcagatgc actctacttg caacgagggt tactacctga ttggtgagga ga - #tcctgtat        420                                                                          - tgtgaactta aaggatcagt agcaatttgg agcggtaagc ccccaatatg tg - #aaaaggtt        480                                                                          - ttgtgtacac cacctccaaa gatcaagaat ggaaaacaca cctttagtga ag - #tagaagta        540                                                                          - tttgagtatc ttgatgcagt aacttatagt tgtgatcctg cacctggacc ag - #atccattt        600                                                                          - tcacttattg gagagagcac gatttattgt ggtgacaatt cagtgtggag tc - #gtgctgct        660                                                                          - ccagagtgta aagtggtcaa atgtcgattt ccagtagtcg aaaatggaaa ac - #agatatca        720                                                                          - ggatttggaa aaaaatttta ctacaaagca acagttatgt ttgaatgcga ta - #agggtttt        780                                                                          - tacctcgatg gcagcgacac aattgtctgt gacagtaaca gtacttggga tc - #ccccagtt        840                                                                          - ccaaagtgtc ttaaagtgtc gacttcttcc actacaaaat ctccagcgtc ca - #gtgcctca        900                                                                          - ggtcctaggc ctacttacaa gcctccagtc tcaaattatc caggatatcc ta - #aacctgag        960                                                                          - gaaggaatac ttgacagttt ggatgtttgg gtcattgctg tgattgttat tg - #ccatagtt       1020                                                                          - gttggagttg cagtaatttg tgttgtcccg tacagatatc ttcaaaggag ga - #agaagaaa       1080                                                                          - ggcacatacc taactgatga gacccacaga gaagtaaaat ttacttctct ct - #ga             1134                                                                          - <210> SEQ ID NO 5                                                           <211> LENGTH: 945                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:  CD46 cDNAON: Description of Artificial                                  delSCR2/subSCR3 variant                                                 - <400> SEQUENCE: 5                                                           - atggagcctc ccggccgccg cgagtgtccc tttccttcct ggcgctttcc tg - #ggttgctt         60                                                                          - ctggcggcca tggtgttgct gctgtactcc ttctccgatg cctgtgagga gc - #caccaaca        120                                                                          - tttgaagcta tggagctcat tggtaaacca aaaccctact atgagattgg tg - #aacgagta        180                                                                          - gattataagt gtaaaaaagg atacttctat atacctcctc ttgccaccca ta - #ctatttgt        240                                                                          - gatcggaatc atacatggct acctgtctca gatgacgcct gttatagggt ct - #tgtgtaca        300                                                                          - ccacctccaa agatcaagaa tggaaaacac acctttagtg aagtagaagt at - #ttgagtat        360                                                                          - cttgatgcag taacttatag ttgtgatcct gcacctggac cagatccatt tt - #cacttatt        420                                                                          - ggagagagca cgatttattg tggtgacaat tcagtgtgga gtcgtgctgc tc - #cagagtgt        480                                                                          - aaagtggtca aatgtcgatt tccagtagtc gaaaatggaa aacagatatc ag - #gatttgga        540                                                                          - aaaaaatttt actacaaagc aacagttatg tttgaatgcg ataagggttt tt - #acctcgat        600                                                                          - ggcagcgaca caattgtctg tgacagtaac agtacttggg atcccccagt tc - #caaagtgt        660                                                                          - cttaaagtgt cgacttcttc cactacaaaa tctccagcgt ccagtgcctc ag - #gtcctagg        720                                                                          - cctacttaca agcctccagt ctcaaattat ccaggatatc ctaaacctga gg - #aaggaata        780                                                                          - cttgacagtt tggatgtttg ggtcattgct gtgattgtta ttgccatagt tg - #ttggagtt        840                                                                          - gcagtaattt gtgttgtccc gtacagatat cttcaaagga ggaagaagaa ag - #gcacatac        900                                                                          #                 945ag agaagtaaaa tttacttctc tctga                           - <210> SEQ ID NO 6                                                           <211> LENGTH: 84                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:  peptideTION: Description of Artificial                            #of SEQ ID NO:1y nucleic acids 280-531                                        - <400> SEQUENCE: 6                                                           - Cys Tyr Arg Glu Thr Cys Pro Tyr Ile Arg As - #p Pro Leu Asn Gly Gln         #                 15                                                          - Ala Val Pro Ala Asn Gly Thr Tyr Glu Phe Gl - #y Tyr Gln Met His Phe         #             30                                                              - Ile Cys Asn Glu Gly Tyr Tyr Leu Ile Gly Gl - #u Glu Ile Leu Tyr Cys         #         45                                                                  - Glu Leu Lys Gly Ser Val Ala Ile Trp Ser Gl - #y Lys Pro Pro Ile Cys         #     60                                                                      - Glu Lys Val Leu Cys Thr Pro Pro Pro Lys Il - #e Lys Asn Gly Lys His         # 80                                                                          - Thr Phe Ser Glu                                                             - <210> SEQ ID NO 7                                                           <211> LENGTH: 38                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:R INFORMATION: Description of Artificial                            #altered CD46cleotide primer for generating                                         construct                                                               - <400> SEQUENCE: 7                                                           #     38           ctcg ttgcagatga agtgcatc                                   - <210> SEQ ID NO 8                                                           <211> LENGTH: 29                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:R INFORMATION: Description of Artificial                            #altered CD46cleotide primer for generating                                         construct                                                               - <400> SEQUENCE: 8                                                           #            29    ttcc gcgccgcgc                                             - <210> SEQ ID NO 9                                                           <211> LENGTH: 41                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:R INFORMATION: Description of Artificial                            #altered CD46cleotide primer for generating                                         construct                                                               - <400> SEQUENCE: 9                                                           #   41             cctc caaagatcaa gaatggaaaa c                               - <210> SEQ ID NO 10                                                          <211> LENGTH: 27                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:R INFORMATION: Description of Artificial                            #altered CD46cleotide primer for generating                                         construct                                                               - <400> SEQUENCE: 10                                                          #             27   tcca aactgtc                                               - <210> SEQ ID NO 11                                                          <211> LENGTH: 26                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:R INFORMATION: Description of Artificial                            #altered CD46cleotide primer for generating                                         construct                                                               - <400> SEQUENCE: 11                                                          #              26  tgct gtgatt                                                - <210> SEQ ID NO 12                                                          <211> LENGTH: 34                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:R INFORMATION: Description of Artificial                            #altered CD46cleotide primer for generating                                         construct                                                               - <400> SEQUENCE: 12                                                          #        34        tata acaggcgtca tctg                                       - <210> SEQ ID NO 13                                                          <211> LENGTH: 34                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:R INFORMATION: Description of Artificial                            #altered CD46cleotide primer for generating                                         construct                                                               - <400> SEQUENCE: 13                                                          #        34        gcca ctttctttac aaag                                       - <210> SEQ ID NO 14                                                          <211> LENGTH: 32                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:R INFORMATION: Description of Artificial                            #altered CD46cleotide primer for generating                                         construct                                                               - <400> SEQUENCE: 14                                                          #          32      cctt ctcacatatt gg                                         - <210> SEQ ID NO 15                                                          <211> LENGTH: 44                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:R INFORMATION: Description of Artificial                            #altered CD46cleotide primer for generating                                         construct                                                               - <400> SEQUENCE: 15                                                          # 44               attg gtgaggagat cctgtattgt gaac                            __________________________________________________________________________

We claim:
 1. A method of increasing production of mammalian CD46 proteinin a host cell, wherein a nucleic acid encoding the CD46 protein has anA and/or T rich region containing more than 60% A and/or T in an exon,said method comprising the steps of:altering the nucleic acid byreducing the amount of A and/or T in said region to less than 60% Aand/or T; transfecting a host cell with said altered nucleic acid, andobtaining expression of said altered nucleic acid under appropriateconditions,wherein the level of CD46 protein production resulting fromexpression of said altered nucleic acid is increased relative to thelevel of CD46 protein production resulting from expression of saidnucleic acid before alteration; and wherein RNA stability of RNAproduced from expression of said altered nucleic acid is notdemonstrably altered relative to the stability of RNA produced fromexpression of said nucleic acid before alteration.
 2. The method claim 1wherein said altered nucleic acid is transfected into said host cell asa component of a recombinant nucleic acid construct.
 3. The method ofclaim 1 wherein said altering of the nucleic acid is by effecting one ormore silent mutations.
 4. The method of claim 1 wherein said altering ofthe nucleic acid is by effecting one or more conservative mutations. 5.The method of claim 1 wherein said altering of the nucleic acid is byinsertion of one or more nucleotides other than A or T.
 6. The method ofclaim 1 wherein said altering of the nucleic acid is by deletion of oneor more nucleotides other than A or T.
 7. The method claim 1 whereinsaid altering of the nucleic acid is by a combination of any two or moreof:a. one or more silent mutations b. one or more conservative mutationsc. insertion of one or more nucleotides other than A or T d. deletion ofA and/or T deletion of A and/or T rich regions.
 8. The method of claim 1wherein said nucleic acid has A and/or T rich regions present in one ormore of its short consensus repeat (SCR) modules.
 9. The method of claim8 wherein said nucleic acid is altered in one or more of its SCRmodules.
 10. The method of claim 1 wherein said altering of the nucleicacid is by deletion of one or more SCR modules or parts thereof.
 11. Themethod of claim 1 wherein said altered nucleic acid comprises a nucleicacid selected from the group consisting of SEQ.ID.No. 2, SEQ.ID.No. 3,SEQ.ID.No. 4 and SEQ.ID.No.
 5. 12. The method of claim 1 wherein saidaltered nucleic acid is transfected into a host cell as a component of arecombinant nucleic acid construct and said recombinant nucleic acidconstruct includes an eukaryotic vector.
 13. The method of claim 1wherein said altered nucleic acid is transfected into a host cell as acomponent of a recombinant nucleic acid construct and said host cell isan eukaryotic host cell.
 14. The method of claim 1 wherein said alterednucleic acid is transfected into a host cell as a component of arecombinant nucleic acid construct and said host cell is COS-7 orCHO-K1.
 15. The method of claim 1 wherein said A and/or T rich regioncontains approximately 78% A and/or T which is reduced or lowered toapproximately 55% by altering the nucleic acid.
 16. The method of claim1 wherein said A and/or T rich region contain approximately 71% A and/orT which is reduced or lowered to approximately 58% by altering thenucleic acid.
 17. A recombinant nucleic acid construct that provides forincreased production of mammalian CD46 protein in a host cell,whereinsaid recombinant nucleic acid construct is formed by altering a nucleicacid encoding the CD46 protein and having an A and/or T rich regioncontaining more than 60% A and/or T by reducing the amount of A and/or Tin said region to less than 60% A and/or T, and wherein RNA stability ofRNA produced from the expression of said altered nucleic acid is notdemonstrably altered relative to the stability of RNA produced fromexpression of said nucleic acid before alteration.
 18. A recombinantnucleic acid construct comprising a nucleic acid selected from the groupconsisting of SEQ.ID.No. 2, SEQ.ID.No. 3, SEQ.ID.No. 4 and SEQ.ID.No. 5.